U.S. patent application number 13/154143 was filed with the patent office on 2014-04-24 for cancer detection markers.
This patent application is currently assigned to Myriad Genetics, Incorporated. The applicant listed for this patent is Ryan Hoff, Jerry Lanchbury, Chris Neff, Zaina Sangale, Mark Skolnick, Kirsten Timms, Susanne Wagner, Hubert Wang. Invention is credited to Ryan Hoff, Jerry Lanchbury, Chris Neff, Zaina Sangale, Mark Skolnick, Kirsten Timms, Susanne Wagner, Hubert Wang.
Application Number | 20140113310 13/154143 |
Document ID | / |
Family ID | 47261958 |
Filed Date | 2014-04-24 |
United States Patent
Application |
20140113310 |
Kind Code |
A9 |
Skolnick; Mark ; et
al. |
April 24, 2014 |
CANCER DETECTION MARKERS
Abstract
Methods and compositions involving molecular markers for the
detection and characterization of cancer in a patient are
provided.
Inventors: |
Skolnick; Mark; (Salt Lake
City, UT) ; Lanchbury; Jerry; (Salt Lake City,
UT) ; Timms; Kirsten; (Salt Lake City, UT) ;
Neff; Chris; (Salt Lake City, UT) ; Hoff; Ryan;
(Salt Lake City, UT) ; Wang; Hubert; (Salt Lake
City, UT) ; Wagner; Susanne; (Salt Lake City, UT)
; Sangale; Zaina; (Salt Lake City, UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Skolnick; Mark
Lanchbury; Jerry
Timms; Kirsten
Neff; Chris
Hoff; Ryan
Wang; Hubert
Wagner; Susanne
Sangale; Zaina |
Salt Lake City
Salt Lake City
Salt Lake City
Salt Lake City
Salt Lake City
Salt Lake City
Salt Lake City
Salt Lake City |
UT
UT
UT
UT
UT
UT
UT
UT |
US
US
US
US
US
US
US
US |
|
|
Assignee: |
Myriad Genetics,
Incorporated
Salt Lake City
UT
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20120309018 A1 |
December 6, 2012 |
|
|
Family ID: |
47261958 |
Appl. No.: |
13/154143 |
Filed: |
June 6, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US09/67029 |
Dec 7, 2009 |
|
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13154143 |
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61120259 |
Dec 5, 2008 |
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61174600 |
May 1, 2009 |
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61175954 |
May 6, 2009 |
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61158975 |
Mar 10, 2009 |
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Current U.S.
Class: |
435/7.4 ;
435/7.92; 436/501 |
Current CPC
Class: |
G01N 33/57434
20130101 |
Class at
Publication: |
435/7.4 ;
436/501; 435/7.92 |
International
Class: |
G01N 33/574 20060101
G01N033/574; G01N 33/577 20060101 G01N033/577 |
Claims
1-26. (canceled)
27. A method of detecting prostate cancer in a patient comprising
determining the level of CD63 in a sample obtained from said
patient; determining the level of at least one additional marker
chosen from the group consisting of: ADAM10, .alpha.V.beta.6
integrin, Caveolin-1, CD147, CD36, CD63, CD81, Claudin-3,
Claudin-4, Desmocollin-1, EGFR, EGFRvIII, EMP-2, EpCAM, ErbB2,
GP1b, HLA-DR, Hsp70, Hsp90, MFG-E8, Rab13, Tissue Factor, CA-125,
CEA, PSA, PSMA, PSAP, CK7, and CK20; and correlating an elevated
level of CD63 and an elevated level of said additional marker in
said sample to an increased likelihood of prostate cancer.
28. The method of claim 27, wherein said additional marker is
EpCAM.
29. The method of claim 27, wherein said additional marker is
PSMA.
30. The method of claim 29, further comprising determining the
level of PSMA.
31. The method of claim 27, further comprising determining the
level of PSA in a sample obtained from said patient and correlating
an elevated level of PSA to the presence of prostate cancer.
32. The method of claim 31, wherein the level of PSA is determined
by the quantification of PSA-bearing exosomes in said sample.
33. The method of claim 27, wherein said sample is chosen from the
group consisting of blood sample, serum sample and plasma
sample.
34. The method of claim 27, wherein said sample is enriched for
exosomes.
35. The method of claim 34, wherein enriching said sample for
exosomes comprises capturing exosomes on a solid surface.
36. The method of claim 27, wherein said level is determined by a
technique chosen from the group consisting of: flow cytometry;
ELISA; electrochemiluminescence ELISA; and IHC.
37. A method of diagnosing prostate cancer in a patient comprising
identifying said patient as at risk of having prostate cancer;
determining the level of CD63 in a sample obtained from said
patient; determining the level of at least one additional marker
chosen from the group consisting of: ADAM10, .alpha.V.beta.6
integrin, Caveolin-1, CD147, CD36, CD63, CD81, Claudin-3,
Claudin-4, Desmocollin-1, EGFR, EGFRvIII, EMP-2, EpCAM, ErbB2,
GP1b, HLA-DR, Hsp70, Hsp90, MFG-E8, Rab13, Tissue Factor, CA-125,
CEA, PSA, PSMA, PSAP, CK7, and CK20; and correlating an elevated
level of CD63 and an elevated level of said additional marker in
said sample to an increased likelihood of prostate cancer.
38. The method of claim 37, wherein said additional marker is
EpCAM.
39. The method of claim 37, wherein said additional marker is
PSMA.
40. The method of claim 38, further comprising determining the
level of PSMA.
41. The method of claim 37, further comprising determining the
level of PSA in a sample obtained from said patient and correlating
an elevated level of PSA to the presence of prostate cancer.
42. The method of claim 37, wherein said sample is chosen from the
group consisting of blood sample, serum sample and plasma
sample.
43. The method of claim 37, wherein identifying said patient as at
risk of having prostate cancer comprises determining that said
patient (a) had a suspicious digital rectal exam, (b) had a
suspicious biopsy, or (c) has elevated levels of PSA.
44. The method of claim 37, wherein said status is determined by a
technique chosen from the group consisting of: flow cytometry;
ELISA; electrochemiluminescence ELISA; and IHC.
45. A kit comprising reagents for the detection of at least three
of the markers chosen from the group consisting of ADAM10,
.alpha.V.beta.6 integrin, Caveolin-1, CD147, CD36, CD63, CD81,
Claudin-3, Claudin-4, Desmocollin-1, EGFR, EGFRvIII, EMP-2, EpCAM,
ErbB2, GP1b, HLA-DR, Hsp70, Hsp90, MFG-E8, Rab13, Tissue Factor,
CA-125, CEA, PSA, PSMA, PSAP, CK7, and CK20.
46. The kit of claim 45, wherein at least one of said reagents is
attached to a solid surface.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is the U.S. national phase of International
Application No. PCT/US2009/067029 filed on Dec. 7, 2009, which
designated the U.S. and claims priority to U.S. Provisional Patent
Application Ser. No. 61/175,954 filed May 6, 2009, U.S. Provisional
Patent Application Ser. No. 61/174,600 filed May 1, 2009, U.S.
Provisional Patent Application Ser. No. 61/158,975 filed Mar. 10,
2009, U.S. Provisional Patent Application Ser. No. 61/120,259 filed
Dec. 5, 2008, the entire contents of each of which are hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention generally relates to a molecular
classification of disease and particularly to molecular markers for
cancer and methods of use thereof.
BACKGROUND OF THE INVENTION
[0003] Cancer is a major health challenge. Nearly 560,000 people
die from cancer annually in the United States alone, representing
almost 23% of all deaths. See American Cancer Society, Cancer Facts
& Figures 2008, 1-2 (2008). Despite recent advances in
molecular and imaging diagnostics, one of the most vexing aspects
of cancer remains early detection. In fact, for certain types of
cancer--e.g., pancreatic adenocarcinoma--detection often occurs so
late as to practically preclude any good prognosis. Thus there is
an urgent need for sensitive methods of detecting cancer.
SUMMARY OF THE INVENTION
[0004] Small extracellular vesicles (e.g., exosomes), or the
markers associated with them, are often found in altered status in
the samples of patients having certain diseases or disease-related
pathologies as compared to samples from healthy individuals. This
is especially true of epithelial cancers (e.g., those of the lung,
colon, breast, prostate, ovaries, endometrium, etc.), diabetic
nephropathy, etc.
[0005] Thus one aspect of the invention provides a method for
detecting cancer in a patient comprising determining the status of
exosomes and/or an exosome-associated marker in a sample obtained
from a patient, wherein an abnormal exosome (and/or
exosome-associated marker) status in the sample indicates the
presence of cancer. In some embodiments status is determined by
determining the level of exosomes or an exosome-associated marker
in the sample. In some embodiments determining the level of
exosomes in the sample comprises measuring the aggregate level of
all exosomes in a sample. In some embodiments status is determined
by measuring the level of exosomes bearing a particular marker.
This may comprise determining the status (e.g., the level) of an
exosome-associated marker. It may also comprise isolating exosomes
from the sample and determining the status (e.g., the level) of an
exosome-associated marker. In some embodiments the
exosome-associated marker is a cancer-marker, a cancer-type marker,
and/or a tissue-type marker. Examples of exosome-associated markers
include those listed in Table 1.
[0006] Some markers are correlated with cancer of a specific type
(e.g., cell type, tissue type, organ, clinical subtype, etc.). Thus
another aspect of the invention provides a method of diagnosing a
cancer in a patient comprising determining the status of an
exosome-associated cancer-type marker in a sample obtained from the
patient, wherein an abnormal status of the marker indicates the
presence of a specific cancer type, or cancer in a specific tissue
or organ, in the patient. In some embodiments the
exosome-associated marker is not necessarily correlated with
cancer, but with a particular tissue type or organ. An example of
such an analysis is given in FIG. 2. In some embodiments exosomes
are isolated and the exosome-associated marker is then analyzed.
Thus one embodiment of the invention provides a method of
diagnosing a cancer in a patient comprising (1) isolating exosomes
from a sample obtained from a patient and (2) determining the
status of a cancer-type marker associated with the exosomes
isolated in (1); wherein an abnormal status of the marker in (2)
indicates the presence of indicates the presence of a specific
cancer type, or cancer in a specific tissue or organ, in the
patient. An example of such an analysis is given in FIG. 2.
Examples of cancer-type markers include some of those listed in
Table 1 (e.g., EGFRvIII, ErbB2).
[0007] Another aspect of the invention provides a method of
screening for cancer in a patient comprising identifying a patient
at risk of having cancer or in need of screening and determining
the status of exosomes and/or an exosome-associated marker in a
sample obtained from the patient, wherein an abnormal status of
exosomes and/or the exosome-associated marker in the sample
indicates the presence of cancer. In some embodiments the patient
is at risk for developing a specific cancer type and the abnormal
status of exosomes and/or the exosome-associated marker indicates
the presence of this specific cancer type.
[0008] Yet another aspect of the invention provides a method of
detecting recurrence in a cancer patient comprising determining the
status of exosomes and/or an exosome-associated marker in a sample
obtained from a patient, wherein an abnormal status of exosomes
and/or the exosome-associated marker in the sample indicates
recurrence.
[0009] Still another aspect of the invention provides a diagnostic
method comprising identifying a patient who is a candidate for
biopsy and determining the status of exosomes and/or an
exosome-associated marker in a sample obtained from the patient,
wherein an abnormal status of exosomes and/or the
exosome-associated marker in the sample indicates a biopsy is
desirable. In some embodiments no abnormal status of exosomes
and/or the exosome-associated marker indicates no biopsy is
necessary.
[0010] One aspect of the invention provides a method of detecting a
specific disease other than cancer (e.g., rheumatoid arthritis,
diabetic nephropathy) comprising determining the status of exosomes
and/or an exosome-associated marker in a sample obtained from a
patient, wherein an abnormal status of exosomes and/or the
exosome-associated marker indicates the patient has the
disease.
[0011] Various techniques can be used to determine the status of
exosomes. Examples of such techniques include, but are not limited
to: flow cytometry; enzyme-linked immunosorbent assay (ELISA) and
its numerous variations (e.g., electrochemiluminescence (ECL));
immunohistochemistry (IHC); etc. or any combination of these.
Exosomes can be isolated by various techniques including, but not
limited to: centrifugation (e.g., ultracentrifugation); physical
filtration; affinity chromatography; contacting a sample with a
solid surface (e.g., plate, well, bead, etc.) containing an
antibody against an exosome-associated marker in order to attach
any exosomes to the surface; etc. or any combination of these.
[0012] Another aspect of the invention provides exosome blocks
comprising fixed (e.g., formalin-fixed) exosomes embedded in some
medium (e.g., paraffin). Such exosome blocks allow for long-term
storage of exosomes and also allow for analysis of the interior of
exosomes.
[0013] These aspects of the invention provide a relatively quick
and inexpensive screen for the presence of cancer. This can be used
as a general population screen (e.g., yearly blood screen) whose
ease, non-invasiveness, patient inclusivity, and sensitivity may
provide vast improvements over existing general screens such as
mammograms, colonoscopies, etc.
[0014] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described
below. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0015] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 illustrates a method, according to the invention, for
detecting cancer generally and also determining the specific cancer
type;
[0017] FIG. 2 illustrates one embodiment of the invention using
various biomarkers to determine which specific cancer is present in
a patient.
[0018] FIGS. 3-7 illustrate differentiating cancer patients from
cancer-free controls by determining exosome status using several
different techniques.
DETAILED DESCRIPTION OF THE INVENTION
[0019] In addition to cells, blood contains various additional
circulating elements. These include, but are not limited to,
cellular debris (e.g., membrane fragments), free protein, protein
aggregates, and small extracellular vesicles. It has been
discovered that markers carried by these elements can be analyzed
in order to detect cancer in patient samples. More specifically, it
has been discovered that capturing these items with a capture
reagent and then detecting them with a detection reagent can
separate cancer patients from controls. For example, small
extracellular vesicles called exosomes have been isolated from
patient samples and their status analyzed by way of
exosome-associated markers so as to differentiate healthy from
diseased individuals. In another example cancer has been detected
by subjecting plasma samples to various antibodies and integrins to
capture circulating elements and analyzing these elements by way of
detection antibodies.
[0020] Cells form several different small vesicles within their
interior that perform various functions. For example, lysosomes
digest excess or worn-out organelles, food particles, and engulfed
viruses or bacteria. Some vesicles are trafficked from the cell
interior to outside the cell. These include exosomes, which are
found in the cell within the multi-vesicular body (MVB) and
released into the extracellular matrix and into various bodily
fluids such as blood, serum, urine, etc. These vesicles often bear,
either inside or on their surface, various biological molecules
such as protein, RNA, DNA, etc. Exosomes are secreted by a variety
of cells, including those involved in immune response (T cells, B
cells, dendritic cells, macrophages) and epithelial cells.
[0021] Exosomes have been determined to be useful markers in
detecting and characterizing cancer. For example, the status of
exosomes in a sample has been found to correlate strongly with the
presence of cancer in the patient from whom the sample was
obtained. More specifically, elevated exosomes and/or
exosome-associated markers in a patient sample indicate the patient
has cancer. See Examples below. Further, exosome-associated markers
can help in characterizing the patient's specific cancer (e.g.,
cell type, tissue type, organ, clinical subtype, etc.).
[0022] Thus one aspect of the invention provides a method for
determining whether an individual has cancer comprising determining
the status of exosomes and/or an exosome-associated marker in a
sample obtained from the individual, wherein an abnormal exosome
(and/or exosome-associated marker) status indicates an increased
likelihood of cancer. As used herein, the "status" of a
biomolecular marker (e.g., exosomes, exosome-associated markers,
etc.) refers to the presence, absence, or extent/level of the
marker or some physical, chemical, or genetic characteristic of the
marker or its expression product(s). Such characteristics include,
but are not limited to, expression level, activity level, structure
(e.g., sequence information, including mutations), copy number,
post-translational modification for proteins (e.g., glycosylation),
etc. These may be assayed directly (e.g., by assaying the
expression level of a particular gene) or determined indirectly
(e.g., assaying the level of marker whose expression level is
correlated to the expression level of the gene of interest).
[0023] In some embodiments determining the status of exosomes
and/or an exosome-associated marker comprises determining their
level in a sample. As used herein in the context of exosomes,
markers, etc., the "level" of something in a sample has its
conventional meaning in the art. Determining a "level" therefore
includes quantitative determinations--e.g., mg/mL, fold change,
etc. Determining a "level" herein also includes more qualitative
determinations--e.g., determining the presence or absence of a
marker or determining whether the level of the marker is "high,"
"low" or even "present" relative to some index value.
[0024] "Abnormal status" means a marker's status in a particular
sample differs from the reference status for that marker (e.g., in
healthy samples or average diseased samples). Examples include
mutated (when the sequence or structure of the marker is analyzed),
elevated, decreased, present, absent, etc. For example, determining
the status of an extracellular protein marker will often include
determining its level in a sample (e.g., on the surface of
exosomes). An abnormal status could be either lower (including
undetectable) or higher levels (including anything non-zero)
compared to the index value in exosome samples from healthy
patients. Another example of abnormal status includes a sequence
(i.e., structural) variation in a protein or gene, such as
EGFRvIII, or in an mRNA, such as KRAS. In this context, a patient
has an "increased likelihood of cancer" if the status of the
relevant marker in the patient's sample is correlated with cancer.
Examples include mutations in particular genes correlated with
cancer, a level of the marker that is closer to some cancer index
value than to a normal index value, a level of the marker that
exceeds some threshold value where exceeding that value is
correlated with cancer, etc. Thus "increased likelihood of cancer"
means a patient with an abnormal status for a marker has a higher
likelihood of cancer than if the patient did not have an abnormal
status.
[0025] An "elevated status" means that one or more of the above
characteristics (e.g., expression) of the marker of interest is
higher than normal levels. Generally this means an increase in the
characteristic (e.g., expression) as compared to an index value.
Conversely a "low status" means that one or more of the above
characteristics (e.g., expression) is lower than normal levels.
Generally this means a decrease in the characteristic (e.g.,
expression) as compared to an index value. In this context, a
"negative status" generally means the characteristic is absent or
undetectable (which would include expression, copy number,
methylation, etc.).
[0026] In some embodiments, the level of a marker is determined
within one or more samples as compared to some index value. Those
skilled in the art will appreciate how to obtain and use an index
value in the methods of the invention. The index value may
represent the average (e.g., mean) level in a plurality of training
patients (e.g., both diseased and healthy patients). For example, a
"cancer index value" can be generated from a plurality of training
patients characterized as having cancer. A "cancer-free index
value" can be generated from a plurality of training patients
defined as not having cancer. Thus, a cancer index value may
represent the average level of a marker (e.g., exosomes,
exosome-associated markers, etc.) in patients having cancer,
whereas a cancer-free index value may represent the average level
of the marker in patients not having cancer. Thus, when the level
of the marker is more similar to the cancer index value than to the
cancer-free index value, then it can be concluded that the patient
has or is likely to have cancer. On the other hand, if the level of
the marker is more similar to the cancer-free index value than to
the cancer index value, then it can be concluded that the patient
does not have, or has no increased likelihood of, cancer.
[0027] Alternatively index values may be determined thusly: In
order to assign patients to risk groups, a threshold value may be
set for the marker. The optimal threshold value is selected based
on the receiver operating characteristic (ROC) curve, which plots
sensitivity vs. (1--specificity). For each increment of the marker
mean (e.g., exosome-associated marker expression), the sensitivity
and specificity of the test is calculated using that value as a
threshold. The actual threshold will be the value that optimizes
these metrics according to the artisan's requirements (e.g., what
degree of sensitivity or specificity is desired, etc.).
[0028] Determining the status of exosomes in a sample may also
comprise assaying some marker whose status itself is correlated
with exosome status. This marker will often be an
exosome-associated marker. "Exosome-associated" marker means a
biomolecule inside or on the surface of an exosome. For example,
exosomes carry cellular biomolecules such as proteins and nucleic
acids (e.g., mRNA, microRNA, etc.) within their lipid membrane.
Since exosomes are formed from membranes of the cell, they also
carry molecules (e.g., cell-surface molecules such as CD63 or
EpCAM) on their surface. Additionally, since some biomolecules bind
those on the surface of exosomes (e.g., antibodies binding an
exosome-surface protein), these biomolecules are also indirectly
"exosome-associated." As discussed below, in some embodiments the
invention need not be limited by the actual physical element that
is measured in the claimed method as long as the status of an
exosome-associated marker such as those listed in Table 1 is
determined. Thus, the invention envisions detecting an
exosome-associated marker even after such marker has been separated
from the exosome with which it is sometimes associated.
[0029] Thus, in one aspect the invention provides a method of
detecting cancer in a patient comprising determining the status of
an exosome-associated marker (e.g., the markers listed in Table 1)
in a sample obtained from the patient, wherein an abnormal status
for such exosome-associated marker indicates an increased
likelihood of cancer. Markers useful in this aspect include those
listed in Table 1, EpCAM, MUC1, CDH1, CK7, PSA, etc.
[0030] It has been determined that the status of each
exosome-associated marker listed in Table 1 (alone or in
combination) is correlated with the presence of cancer. See
Examples below.
TABLE-US-00001 TABLE 1 Marker Name/Symbol Entrez GeneId No. ADAM10
102 .alpha.V.beta.6 integrin 3678, 3694 Caveolin-1 857 CD147 382
CD36 948 CD63 967 CD81 975 Claudin-3 1365 Claudin-4 1364
Desmocollin-1 1823 EGFR 1956 EGFRvIII 1956 EMP-2 2013 EpCAM 4072
ErbB2 2064 GP1b 2811 HLA-DR Various Hsp70 3308 Hsp90 3320 MFG-E8
4240 Rab13 5872 Coagulation factor III 2152
[0031] Thus in some embodiments the invention provides a method of
detecting cancer comprising determining the status of at least one
marker chosen from the group consisting of: ADAM10,
a.alpha.V.beta.6 integrin, Caveolin-1, CD147, CD36, CD63, CD81,
Claudin-3, Claudin-4, Desmocollin-1, EGFR, EGFRvIII, EMP-2, EpCAM,
ErbB2, GP1b, HLA-DR, Hsp70, Hsp90, MFG-E8, Rab13, and Tissue
Factor, wherein an abnormal status indicates an increased
likelihood of cancer.
[0032] Patient blood samples are complex mixtures of circulating
elements including, but not limited to, cellular debris (e.g.,
membrane fragments), free protein, protein aggregates, and small
extracellular vesicles (e.g., exosomes). In some embodiments the
patient blood sample is enriched for exosomes and/or
exosome-associated markers. In other words the sample is enriched
to remove cells and individual proteins. "Sample" as used herein
refers to any biological specimen, including any tissue or fluid,
that can be obtained from, or derived from a specimen obtained
from, a human subject. Such samples include, healthy or tumor
tissue, bodily fluids, waste matter (e.g., urine, stool), etc. In
some embodiments the sample is blood or any substance derived
therefrom--e.g., serum or plasma. The process of extracting plasma
from blood, by removing the blood cells, helps to enrich for
exosomes and/or exosomes-associated markers. Further purifying
plasma to serum, by removing fibrinogen and other clotting factors,
further enriches the sample. Additional enrichment/purification may
be performed by various techniques discussed in more detail below
including, but not limited to, ultracentrifugation, filtration,
affinity chromatography, antibody capture, cell-sorting, etc.
Though the methods of the invention can differentiate cancer
patients from controls in both plasma and serum samples, it has
been discovered that plasma samples yield better (i.e., clearer)
differentiation. See FIG. 4.
[0033] Thus in one aspect the invention provides a method of
detecting cancer comprising enriching a sample for exosomes and
determining the status of the exosomes and/or an exosome-associated
marker, wherein an abnormal status for exosomes and/or the
exosome-associated marker indicates an increased likelihood of
cancer. In some embodiments the exosome-associated marker is chosen
from those listed in Table 1. In some embodiments the sample is
blood plasma and the sample is enriched by a technique chosen from
the group consisting of ultracentrifugation, filtration, affinity
chromatography, antibody capture and cell-sorting. In some
embodiments the sample is enriched using ultracentrifugation and
the resulting exosomes (or other circulating elements) are coated
onto a solid surface for subsequent detection (see Examples 2, 4
& 5). In some embodiments the sample is enriched using a
capture antibody specific for one of the markers listed in Table 1
and the exosomes and/or exosome-associated markers are detected
using an antibody specific for another marker listed in Table
1.
[0034] Though the inventors have devised assays aimed at measuring
exosomes and exosome-associated markers from patient blood, plasma
and serum, some aspects of the invention need not be limited to the
particular circulating element that is actually measured.
Specifically, in some examples the inventors have attempted to
isolate/purify exosomes from blood (or plasma or serum) samples
using antibody or integrin capture, ultracentrifugation and/or
filtration, followed by detection (e.g., via ELISA, ECL, flow
cytometry, Bradford protein measurement, etc.). See Examples 1, 2
& 5. In some cases, however, it is ultimately the choice of
markers and/or assays that has enabled cancer detection regardless
of whether exosomes themselves (or some other circulating element)
have been directly measured. Thus, in some examples no significant
attempt has been made at isolating or purifying exosomes other than
using distinct capture and detection reagents (e.g., anti-CD63
antibody for capture and anti-Claudin-3 antibody for detection).
See Example 4. Though not wishing to be bound by theory, it is
thought that using distinct capture and detection reagents will not
detect single circulating proteins and, in the case of plasma, will
not detect whole cells. In the case of serum, clotting factors have
further been eliminated. Such an assay should instead detect
exosomes or small membrane fragments, protein complexes, etc.
containing exosome-associated markers (e.g., those listed in Table
1).
[0035] Thus one aspect of the invention provides a method of
detecting cancer comprising determining the status of at least two
markers chosen from those listed in Table 1 in a patient sample,
wherein an abnormal status for at least one of the markers
indicates an increased likelihood of cancer. In some embodiments
one of the markers is used to capture circulating elements from the
sample while another of the markers is used to detect (e.g.,
quantify) these elements. In some embodiments the level of the
markers is determined using an antibody specific for each marker.
In some embodiments the status of the capture marker is its
presence or absence, which is determined by attaching the capture
marker to a solid surface via a capture reagent (e.g., antibody,
nucleic acid probe, etc.), while the status of the other marker is
its level as determined via a detection reagent (e.g., antibody,
nucleic acid probe, etc.). The markers listed in Table 1 can be
used in combination in this way, including but not limited to the
combinations described in Example 4.
[0036] In some embodiments the exosome-associated marker is a
cancer marker. As used herein, "cancer marker" means a biomarker
whose status is correlated with the presence of cancer. There need
not be a strong correlation between the biomarker and any specific
cancer type. Examples include carcinoembryonic antigen (CEA) and
epithelial membrane antigen (EMA). Another example includes EpCAM,
which is hyperglycosylated in carcinoma tissue as compared to
corresponding normal epithelial tissue. See, e.g., Munz et al.,
FRONT BIOSCI. (2008) 13:5195-5201. Thus some embodiments provide a
method of detecting cancer comprising providing a sample obtained
from a patient and determining the level of exosomes having a
general cancer marker (e.g., hyperglycosylated EpCAM), wherein an
increased level of these exosomes indicates an increased likelihood
of cancer.
[0037] Some markers are correlated with cancer of a specific type
(e.g., cell type, tissue type, organ, clinical subtype, etc.). For
example, certain biomarkers can be used to differentiate exosomes
derived from epithelial cells from other exosomes, e.g., immune
cell-derived. One particularly useful marker in detecting,
quantitating, collecting, and/or analyzing epithelial exosomes is
EpCAM, a protein expressed on the surface of many epithelial cells.
Other epithelial markers useful in the invention include, but are
not limited to, CDH1 (cadherin 1, type 1, E-cadherin [epithelial])
and cytokeratin 7 (CK7 or KRT7). Elevated levels of epithelial
exosomes are often found in individuals having an altered
physiological condition. For example, pregnant women show higher
levels of epithelial exosomes in their serum, as do diabetic
patients suffering from nephropathy. Further, epithelial exosomes
are more abundant in samples from cancer patients than in those
from cancer-free controls.
[0038] Thus another aspect of the invention provides a method of
diagnosing a cancer in a patient comprising determining the status
of an exosome-associated cancer-type marker in a sample obtained
from the patient, wherein an abnormal status of the marker
indicates the presence of a specific cancer type, or cancer in a
specific tissue or organ, in the patient. In some embodiments
exosomes are isolated to some degree and the cancer-specific
exosome-associated marker is then analyzed. Thus one embodiment of
the invention provides a method of diagnosing a cancer in a patient
comprising (1) isolating exosomes from a sample obtained from a
patient and (2) determining the status of a cancer-type marker
associated with the exosomes isolated in (1); wherein an abnormal
status of the marker in (2) indicates the presence of indicates the
presence of a specific cancer type, or cancer in a specific tissue
or organ, in the patient. Examples of cancer-type markers include
some of those listed in Table 1 (e.g., EGFRvIII, ErbB2). An example
of such an analysis is given in FIG. 2.
[0039] As used herein, "cancer type" means a cancer in or
originating from a particular tissue or organ and/or a cancer with
a particular molecular or clinical feature. Often, the specificity
of the "cancer type" varies with the application, including tissue
type (e.g., squamous versus cuboidal), organ type (e.g., breast
versus lung), and clinical subtype (e.g., triple-negative breast
cancer). For example, finding certain markers (e.g., surfactant
proteins B [SFTPB] and C [SFTPC]) on exosomes derived from a tumor
can indicate that it is a lung tumor. See, e.g., Johnson et al., J.
BIOL. CHEM. (2001) 276:14658-14664. Breast cancer cells can show
overexpression of HER2, which in turn correlates with a particular
clinically-defined subtype of breast cancer. Knowing that the
patient has breast cancer will generally prompt a physician to
treat in a particular way (e.g., surgery, hormone therapy, and
optional adjuvant chemotherapy), while the additional knowledge
that the patient has HER2-overexpressing breast cancer can prompt
further personalization of that treatment (e.g., adding
trastuzumab).
[0040] The present invention provides various methods of
determining what cancer type a patient has. For example, cancer
type may be determined by more traditional diagnostic methods such
as imaging (e.g., CAT scan, MRI, X-ray, etc.), physical examination
(e.g., digital rectal examination--DRE--in prostate cancer),
biopsy, etc. Thus, one aspect of the invention provides a method of
determining whether a patient has a specific cancer type,
comprising determining the status of exosomes and/or an
exosome-associated marker in a sample obtained from the patient,
wherein an abnormal status of such exosomes and/or the
exosome-associated marker indicates the presence of cancer, and
performing a physical examination, imaging test, or biopsy to
determine the specific cancer type.
[0041] Alternatively, molecular diagnostics may give cancer-type
specificity. For example, the cancer marker in the above
embodiments may simultaneously be a cancer-type marker. As used
herein, "cancer-type marker" means a biomarker whose status is
correlated with the presence of a specific cancer type. One example
is prostate-specific antigen (PSA), where a status of elevated
levels is correlated with prostate cancer. Thus one embodiment
provides the quantification of PSA-bearing exosomes as a substitute
for measuring PSA in the serum, which may be a more clinically
accurate measure of cancer-relevant PSA. Another antigen of
particular interest is CA-125. It is frequently expressed in
ovarian tumors. It is also expressed at lower levels in normal
individual's serum, but since such individuals either don't have
exosomes or have far fewer of them, comparing CA-125-bearing
exosomes in individuals with tumors compared to controls could be
much more effective than comparing levels of CA-125 free in serum.
CA-125 is somewhat non-specific for tumor type in that it is
expressed in gynecological cancers other than ovarian and in colon
cancer. Thus elevated levels of CA-125-bearing exosomes could
indicate the value of more expensive and invasive tests such as an
MRI of the abdominal/pelvic region, a pelvic exam, a colonoscopy
and possibly biopsies. Yet another example is EGFRvIII, whose
expression is correlated with a particular form of glioblastoma
(i.e., particular mutated EGFR status is correlated with a specific
clinical subtype of glioblastoma). Those skilled in the art are
familiar with additional examples (e.g., HER2, EGFR, KRAS,
etc.).
[0042] Another option to specify the cancer type is by determining
that the patient has cancer and then, through the same or a
subsequent assay, determining the tissue of origin using a
tissue-specific marker. As used herein, a "tissue-specific marker"
is a biomarker whose status is correlated with a specific tissue or
organ type, though not necessarily with cancer. Several examples of
such tissue-specific markers, as well as the tissues and/or organs
with which they are correlated, are given in FIG. 2. This
additional marker is generally, though not necessarily,
exosome-associated.
[0043] Thus, one aspect of the invention provides a method of
determining whether a patient has a specific cancer type,
comprising determining the status of exosomes and/or an
exosome-associated marker (e.g., those listed in Table 1) in a
sample obtained from the patient and determining the status of at
least one exosome-associated tissue-specific marker in a sample
obtained from the patient, wherein an elevated level of exosomes
and/or the exosome-associated marker indicates the patient has
cancer and wherein a particular status of the exosome-associated
tissue-specific marker indicates the patient has a specific cancer
type. In the context of tissue-type markers, status will often,
though not necessarily, mean level (i.e., presence, absence and/or
amount). Often a particular biomolecule is exclusively or
predominantly found in or produced by a particular tissue type or
organ. Examples include EpCAM (epithelial tissue), SFTPB (lung
epithelium), PSA (prostate tissue), etc. As another example, GPA33
(glycoprotein A33 [transmembrane]) is expressed in the epithelium
of the colon and small intestine. See, e.g., Heath et al., PROC.
NATL. ACAD. SCI. USA (1997) 94:469-474. Thus, one skilled in the
art could, according to the present invention, determine the level
of exosomes (e.g., by determining the level of one or more of the
exosome-associated markers listed in Table 1) and also determine
whether or to what extent (e.g., what proportion of) such exosomes
also bear the GPA33 antigen. If exosome levels are elevated and
some significant proportion of these exosomes also bear the GPA33
antigen, then the patient has colon cancer. In other embodiments
for example, determining that exosome levels are high and PSA is
present (particular PSA "status") will indicate the patient has
prostate cancer. Other times, particular biomolecules are
upregulated in specific tissue types or organs (i.e., found in
higher levels in some tissues or organs as compared to others).
[0044] There is a large and ever growing catalog of
exosome-associated, cancer-type, and tissue-specific markers.
Further, those skilled in the art will appreciate that these
categories are not mutually exclusive (e.g., an exosome-associated
marker may also be tissue-specific). Thus one aspect of the
invention provides methods comprising determining the status of a
panel of markers. In some embodiments, the invention provides a
method of determining whether a patient has cancer comprising
determining the status of a panel of exosome-associated markers in
a sample obtained from the patient, wherein a particular status of
the panel of markers indicates the patient has cancer. In some
embodiments, the invention provides a method of determining whether
a patient has a specific cancer type, comprising determining the
status of a panel of cancer-type markers in a sample obtained from
the patient, wherein a particular status of the panel of markers
indicates the patient has a specific cancer type. In yet other
embodiments, the invention provides a method of determining whether
a patient has a specific cancer type, comprising determining the
status of exosomes and/or an exosome-associated marker in a sample
obtained from the patient and determining the status of a panel of
tissue-specific markers in a sample obtained from the patient,
wherein an abnormal status of exosomes and/or the
exosome-associated marker indicates the patient has cancer and
wherein a particular status of the panel of markers indicates the
patient has a specific cancer type (see, e.g., FIG. 2).
[0045] In some embodiments the panel of markers comprises 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,
35, 40, 45, 50, 60, 70, 80, 90, 100, or more markers. The markers
may be any exosome-associated markers, including cancer-type
markers, or tissue-type markers or a combination of these. In some
embodiments the panel of markers comprises two or more markers
listed in Table 1. In some embodiments the panel of markers
comprises two or more markers shown in FIG. 2, wherein the presence
or absence (or abnormal status) of specific markers indicates,
according to the flowcharts in FIG. 2, the patient has cancer of a
specific type. In some embodiments the panel comprises at least two
markers chosen from those listed in Table 1.
[0046] In further embodiments the status of individual markers in
the panel is tested in a certain order in order to narrow down
which specific cancer type is present. One example is illustrated
in FIG. 2A-2D. Specifically, when elevated exosome levels are found
in a patient's sample, one may also test the sample for the status
of cytokeratin 7 (CK7) and cytokeratin 20 (CK20) followed by
various other markers. If both CK7 and CK20 are absent as in FIG.
2A [110], then PSA, PSAP, PSMA, Hep Par 1, AFP, CAM 5.2, CD10,
Vimentin, RCC, and EMA (or any combination thereof or any single
marker) may be tested [210] to determine the specific organ/tissue
of origin. If PSA, PSAP, and/or PSMA are found, then the cancer is
prostate adenocarcinoma [310]. If Hep Par 1, AFP, and/or CAM 5.2
are present, then the cancer is hepatocellular carcinoma [311]. If
CD10, Vimentin, RCC, and/or EMA are present, then the cancer is
renal cell carcinoma (clear cell type) [312].
[0047] If CK7 is absent and CK20 is present as in FIG. 2B [120],
then Ae1/3, CAM 5.2, CK19, CEA (polyclonal), and EMA (or any
combination thereof or any single marker) may be tested [220] to
confirm that the cancer is colon adenocarcinoma. If any of these
markers is found, then the cancer is colon adenocarcinoma [320].
Imaging and/or endoscopy may be performed [420] either in place of
the additional marker tests [320] or as an additional
confirmation.
[0048] If CK7 is present and CK20 is absent as in FIG. 2C [130],
then PSA, PSAP, PSMA, Thyroglobulin, Calictonin, HER2, GCDFP-15,
Chromogranin, Synaptophysin, CD56, (NCAM), Leu7, CK5/6, CEA,
Mucicarmine, B72.3, Leu, M1, (CD15), Calretinin, HBME-1, Mesothelin
and Vimentin (or any combination thereof or any single marker) may
be tested [230] to determine the specific organ/tissue of origin.
If PSA, PSAP, and/or PSMA are found, then the cancer is prostate
cancer [330]. If Thyroglobulin and/or Calictonin are present, then
the cancer is thyroid cancer [331]. If HER2 and/or GCDFP-15 are
found, then the cancer is breast cancer [332]. If Chromogranin,
Synaptophysin, CD56, (NCAM), and/or Leu7 are found, then the cancer
is small cell/neuroendocrine carcinoma of the lung [336]. If CK5/6
is found, then the cancer may be squamous cell carcinoma of the
lung [337] (diagnosis may be confirmed by imaging [430]). CEA,
Mucicarmine, B72.3, and/or Leu M1 (CD 15) are found, then the
cancer may be adenocarcinoma of the lung [338] (diagnosis may be
confirmed by imaging [430]). If Calretinin, HBME-1, CK5/6, and/or
Mesothelin are found, then the cancer may be mesothelioma [333] (if
the only marker found is CK5/6, imaging [430] may be necessary). If
Vimentin is found, then the cancer is endometrial cancer [334]. If
CK5/6 and/or CEA are found, then the cancer may be cervical cancer
[332] (confirmation, e.g., by pap smear, may be necessary since
these markers are also expressed by other CK7+/CK20- tissue
types).
[0049] If CK7 and CK20 are both present as in FIG. 2D [140], then
CA-125, Mesothelin, 34.beta.E12, Villin, Uroplakin III, and/or CD10
(or any combination thereof or any single marker) may be tested
[240] to determine the specific organ/tissue of origin. If CA-125
and/or Mesothelin are found, then the cancer may be ovarian
carcinoma [340] (confirmation, e.g., by imaging, may be necessary
since CA-125 is also expressed in other CK7+/CK20+ tissues). If
34.beta.E12, Villin, and/or CA-125 are present, then the cancer may
be cholangio carcinoma (bile duct cancer) [341] (confirmation,
e.g., by imaging, may be necessary since CA-125 is also expressed
in other CK7+/CK20+ tissues). If Uroplakin III is found, then the
cancer is urothelial carcinoma [342]. If CD10 is found, then the
cancer is papillary-type renal cell carcinoma [343]. If no marker
is found, then the cancer may be chromophobe renal cell carcinoma
[344] (diagnosis may be confirmed microscopically).
[0050] In some patients, a diagnosis of the desired specificity
requires more than simply determining the status of exosomes and/or
an exosome-associated marker. Many physiological conditions other
than cancer are characterized by increased exosome secretion,
including rheumatoid arthritis. For example, as mentioned above,
epithelial exosomes are elevated in pregnant women and in patients
with diabetic nephropathy. In many cases, therefore, alternative
conditions must be ruled out before a diagnosis of cancer can be
made. This may require additional tests specific for the given
alternative condition, e.g., a pregnancy test.
[0051] Thus, one aspect of the invention provides a method for
determining whether a patient has cancer comprising determining the
status of exosomes and/or an exosomes-associated marker in a sample
from this patient and determining whether the patient has a
non-cancerous condition characterized by increased exosome
secretion, wherein an abnormal status of exosomes (e.g.,
EpCAM-bearing exosomes, CD63-bearing exosomes, etc.) and/or the
exosome-associated marker and the absence of any non-cancerous
condition characterized by increased exosome secretion indicates
the patient has cancer. Examples of how one might determine whether
a patient has a noncancerous condition characterized by increased
exosome secretion include, but are not limited to, determining
whether the patient is pregnant (e.g., by a pregnancy test),
determining whether the patient is diabetic (or determining whether
the patient shows signs of diabetic nephropathy), etc.
[0052] Alternatively, one may rule out other conditions by
differentiating tumor-derived exosomes, referred to herein
variously as oncosomes or cancer-derived exosomes, from exosomes
derived from some other cell. Such oncosomes are especially useful
according to the present invention in detecting, classifying and
monitoring cancer since they are particularly prevalent in
epithelial cancers such as those of the lung, colon, breast,
prostate, ovaries, endometrium, etc. One way to differentiate
oncosomes from noncancerous exosomes is by determining the status
of some exosome-associated biomarker. Generally, the methods of the
invention will involve determining whether a particular marker has
an abnormal status, when such abnormal status is somehow associated
with cancer cells. Determining whether a marker's status is
abnormal in a sample will generally comprise comparing the marker's
status in the patient's sample with the marker's index or reference
status (e.g., index values discussed above) in an average, normal,
or healthy patient's sample.
[0053] Another aspect of the invention provides a method of
screening for cancer in a patient comprising identifying a patient
at risk of having, or in need of screening for, cancer and
determining the status of exosomes and/or an exosomes-associated
marker in a sample obtained from the patient, wherein an abnormal
status of exosomes and/or an exosomes-associated marker in the
sample indicates the presence of cancer. Patients may be identified
as at risk of having, or in need of screening for, cancer in a
variety of ways and based on numerous clinical and/or molecular
characteristics. One class of patients at risk of having cancer and
in need of screening is those patients known to carry a germline
deleterious mutation in a tumor suppressor gene. Examples include,
but are not limited to, BRCA1, BRCA2, PTEN, p16, MLH1, MSH6, APC,
MYH, etc. In such patients, cancer-type specificity is often less
crucial since, for example, a BRCA1-mutant patient whose exosome
levels indicate cancer would be expected have breast or ovarian
cancer rather than some other type of cancer. However, if desired,
additional tests to determine the type of cancer may be performed
as discussed above. Age and environmental factors may also define
at-risk patients in need of screening. For example, prostate cancer
is overwhelmingly found in men over 50 years of age. Thus, men over
50 may be considered patients at risk of having, or in need of
screening for, prostate cancer. The relatively non-invasive nature
of blood, plasma, or serum detection (i.e., simple blood draw)
makes such widespread screening attractive and practical.
[0054] Thus in some embodiments the invention provides a method of
detecting cancer comprising identifying a patient having a mutation
in a gene selected from the group consisting of BRCA1, BRCA2, PTEN,
p16, MLH1, MSH6, APC, and MYH; and determining the status of
exosomes and/or an exosomes-associated marker in a sample obtained
from the patient; wherein an abnormal status of exosomes and/or an
exosomes-associated marker in the sample indicates the presence of
cancer. In some such embodiments the method further comprises
additional tests to determine/confirm which type of cancer is
present.
[0055] Yet another aspect of the invention provides a method of
detecting recurrence in a cancer patient comprising providing a
sample obtained from the patient and determining the status of
exosomes and/or an exosomes-associated marker in the sample,
wherein an abnormal status of exosomes and/or an
exosomes-associated marker in the sample indicates recurrence.
Because it is difficult to remove or kill all cancerous cells, one
of the main challenges in cancer treatment is making sure a cancer
removed by surgery and/or treated with drugs has not returned. Thus
this aspect of the invention is particularly useful in monitoring
cancer patients following treatment. Much like the at-risk patients
discussed above, cancer-type specificity is not crucial. If a lung
cancer patient is found to have increased exosome levels in his
blood, plasma or serum several months or years after treatment,
then the new cancer is likely to be a return of the former lung
cancer. As above, in some embodiments further testing (e.g.,
imaging) to confirm the type of cancer or to characterize the
cancer (e.g., stage) is encompassed by the invention. In some
embodiments exosome levels are measured soon after treatment (e.g.,
to determine a post-treatment baseline) and then monitored at
regular intervals there after in order to catch any significant
increase (e.g., from this baseline).
[0056] Still another aspect of the invention provides a method
comprising identifying a patient who is a candidate for biopsy and
determining the status of exosomes and/or an exosomes-associated
marker in a sample obtained from the patient, wherein an abnormal
status of exosomes and/or an exosomes-associated marker in the
sample indicates a biopsy is desirable. Biopsies are often
expensive, painful, and time-consuming and, furthermore, most
biopsies are negative for cancer. Thus this aspect of the invention
provides an effective tool which could allow one to know whether a
biopsy is required (e.g., after PSA, mammography, etc.). As used
herein, "candidate for biopsy" refers to a patient suspected of
having cancer, for whom a biopsy could be expected to confirm the
presence of such cancer. For example, if a patient's mammography
indicates the presence of an abnormal mass, methods according to
this aspect of the invention can help determine whether the mass is
likely to be cancerous and thus determine whether the discomfort
and expense of a biopsy is warranted.
[0057] As mentioned above, some embodiments of the invention
involve exosome analysis combined with more traditional diagnostic
techniques. For example, physical examination (e.g., digital rectal
exam for prostate cancer), imaging (e.g., mammography), and/or
biopsy may be used to confirm a diagnosis indicated by exosome
analysis according to the invention. Alternatively, such techniques
may be combined with exosome analysis to yield a more comprehensive
diagnosis. As an illustrative example, an exosome screen may
indicate the presence of cancer in a patient and these exosomes may
be found to be CK7+/CK20- and have the marker CK5/6 associated with
them. One may not be able to conclusively determine based solely on
this information whether the cancer is squamous cell carcinoma of
the lung, cervical cancer, or mesothelioma at some unknown organ
(see FIG. 2C). Thus, a physician may take the further step of
imaging to pinpoint the location of the cancer (e.g., in or near
the lung). The physician may further perform a biopsy to determine
whether the cancer is squamous cell carcinoma of the lung or cancer
of the mesothelial lining of the lung.
[0058] Various techniques can be used to enrich for exosomes and/or
exosome-associated markers and to determine the status of exosomes
and/or exosome-associated markers in a sample. Examples of
enrichment techniques include, but are not limited to: flow
cytometry (i.e., cell sorting); centrifugation (e.g.,
ultracentrifugation); physical filtration; affinity chromatography;
contacting a sample with a solid surface (e.g., plate, well, bead,
etc.) containing an antibody against an exosome-associated marker
in order to capture any exosomes on the surface; etc. or any
combination of these. Example 5 describes one technique using
ultracentrifugation combined with filtration and ECL to enrich a
sample. As used herein, "ultracentrifugation" has its conventional
meaning in the art. In some embodiments ultracentrifugation
comprises spinning the sample at least 20,000.times.g, at least
30,000.times.g, at least 40,000.times.g, at least 50,000.times.g,
at least 75,000.times.g, at least 100,000.times.g, at least
150,000.times.g, at least 250,000.times.g, at least
500,000.times.g, at least 750,000.times.g, or at least
1,000,000.times.g. Those skilled in the art will appreciate that
several of the variables in the Examples can be adjusted while
still allowing for enrichment. Various suitable sizes and types of
filtration may be used, including filtering to elute exosome-sized
particles in the sample (e.g., >100 nm, 150 nm, 200 nm, 250 nm),
filtering to capture exosome-sized particles (e.g., <50 nm, 40
nm, 30 nm, 20 nm, 10 nm), or both combined.
[0059] Various techniques can be used to determine the status of
exosomes and/or an exosome-associated marker in a patient sample
including, but are not limited to: flow cytometry; enzyme-linked
immunosorbent assay (ELISA) and its numerous variations (e.g.,
electrochemiluminescence or "ECL"); immunohistochemistry (IHC);
etc. or any combination of these. As can be seen, determining the
status of exosomes and/or exosome-associate markers will often also
involve some level of enrichment.
[0060] For example, flow cytometry can both sort exosomes (i.e.,
separate exosomes of interest out of the sample milieu) and
quantitate them, as shown in Example 1. Flow cytometry is
well-suited to the methods of the invention since multiple markers
may be assayed at once by analyzing a panel of antigens by cell
sorting using antibodies to each antigen and counting on a
multichannel sorter. Thus the invention provides a method of
quantitating exosomes comprising isolating exosomes from a patient
sample and counting the exosomes using flow cytometry. In some
embodiments the isolation and counting may be done simultaneously,
such as by using fluorescent-activated cell sorting (FACS) adapted
for use with exosomes.
[0061] Similarly, ECL ELISA can enrich a sample for exosomes and/or
exosome-associated markers by capturing these on a solid surface
(using, e.g., integrins, antibodies, etc.) and can then determine
their level by way of a separate detection reagent (e.g.,
integrins, antibodies, etc.). See Example 4. Thus the invention
provides a method of detecting cancer comprising fixing exosomes
and/or an exosome-associated marker to a solid surface and
determining the level of at least one exosome-associated marker,
wherein an abnormal status of the exosome-associated marker
indicates an increased likelihood of cancer. In some embodiments
the exosomes and/or exosome-associated markers are fixed to the
solid surface by way of a linker. In some embodiments the linker
specifically binds exosomes and/or an exosome-associated marker
(e.g., integrins, antibodies such as anti-CD63, anti-CD36, or any
antibody against any marker listed in Table 1, etc.).
Alternatively, the solid surface can be "sticky"--i.e., the surface
may be made of a material that itself binds exosomes and/or
exosome-associated markers (e.g., latex, graphite).
[0062] In some embodiments IHC is used to determine the status of
exosomes and/or an exosome-associated marker. This will often be in
connection with exosome blocks. Thus the invention provides a
method of detecting cancer comprising determining the status of an
exosome-associated marker by contacting an exosome block sample
from a patient with at least one antibody that specifically binds
the marker, wherein an abnormal status for the marker indicates an
increased likelihood of cancer.
[0063] Though much of the preceding discussion has focused on
exosome-surface markers, the invention is not so limited as
exosome-associated markers may also be found within an exosome's
interior. Examples include proteins, mRNA, microRNA, DNA, etc.
Various techniques exist to analyze the interior exosome-associated
markers. To analyze protein markers, immunological techniques are
available including, but no limited to, intraexosomal staining flow
cytometry (see Example 1 below), ELISA (see Example 2 below), IHC
(e.g., using exosome blocks such as in Example 3 below), etc. To
analyze a nucleic acid marker, the exosomes may be solubilized to
release their contents and nucleic acids of interest may be
quantitated (e.g., qPCR, microarray), genotyped (e.g.,
TaqMan.RTM.), sequenced, etc. Alternatively, fluorescence in situ
hybridization (FISH) may be adapted to analyzing exosome-associated
nucleic acid markers (especially using exosome blocks).
[0064] Another aspect of the invention provides exosome blocks.
These blocks may be prepared in a manner similar to formalin-fixed
paraffin-embedded (FFPE) tissue samples, the main difference being
that isolated exosomes rather than excised tissue form the basis of
exosome blocks. Pathologists routinely make cell pellets from
formalin-fixed body fluid samples and embed them in paraffin to
produce a cell block. Likewise, one may take the fixed, pelleted
exosomes and embed them in paraffin to make an exosome block. This
tissue block can then be sliced (e.g., with a microtome) and
sections put on slides, or a tissue microarray can be constructed.
The interior of many of the exosomes will be exposed when the
exosome block is sliced, and any analysis aimed at interior
exosome-associated markers (e.g., IHC with cytoplasmic antibodies)
can be performed to, e.g., determine the tissue of origin of the
exosome pellet. Thus the invention provides an exosome block
comprising formalin-fixed exosomes embedded in paraffin.
[0065] The results of these and any other analyses according to the
invention will often be recorded and communicated to physicians,
genetic counselors and/or patients (or other interested parties
such as researchers) in a transmittable form that can be
communicated or transmitted to any of the above parties. Thus one
aspect of the invention provides a method comprising determining
the status of exosomes and/or an exosome-associated marker in a
sample obtained from a patient, recording and/or communicating
whether the status is abnormal, and recording and/or communicating
that an abnormal status indicates the presence of cancer. In one
embodiment the method comprises determining the level of exosomes
in a sample obtained from a patient and, if the level of exosomes
is increased, recording and/or communicating that the patient has
an increased likelihood of cancer.
[0066] Transmittable forms for communicating the results of a test
can vary and can be tangible or intangible. The results can be
embodied in descriptive statements, diagrams, photographs, charts,
images or any other visual forms. For example, graphs showing
exosome level information can be used in explaining the results.
Diagrams showing such information for additional markers are also
useful in indicating some testing results. The statements and
visual forms can be recorded on a tangible medium such as papers,
computer readable media such as floppy disks, compact disks, etc.,
or on an intangible medium, e.g., an electronic medium in the form
of email or website on Internet or intranet. In addition, results
can also be recorded in a sound form and transmitted through any
suitable medium, e.g., analog or digital cable lines, fiber optic
cables, etc., via telephone, facsimile, wireless mobile phone,
internet phone and the like.
[0067] Thus, the information and data on a test result can be
produced anywhere in the world and transmitted to a different
location. As an illustrative example, when an exosome level assay
is conducted outside the United States, the information and data on
a test result may be recorded or generated, cast in a transmittable
form as described above, and then imported into the United States.
Accordingly, the present invention also encompasses a method for
producing a transmittable form of information on exosome (and/or
exosome-associated marker) status for at least one patient sample.
The method comprises the steps of (1) determining exosome (and/or
exosome-associated marker) status according to the methods of the
present invention; and (2) embodying the result of the determining
step in a transmittable form. The transmittable form is the product
of such a method.
[0068] Techniques for analyzing such expression, activity, and/or
sequence data (indeed any data obtained according to the invention)
will often be implemented using hardware, software or a combination
thereof in one or more computer systems or other processing systems
capable of effectuating such analysis.
[0069] One aspect of the invention provides methods of treating a
patient comprising determining the status of exosomes and/or an
exosomes-associated marker as discussed above. The treatment
methods of the invention generally further comprise some action
based on the exosome (and/or exosome-associated marker) status
determination. Examples of actions based on the exosome
determination include recommending a biopsy; prescribing a
particular treatment; administering a particular therapeutic
composition; prescribing, recommending, or performing surgery;
monitoring for more signs of malignancy (e.g., recommending an
additional test for cancer), etc. Other actions (such as
communicating, e.g., a particular diagnosis or prognosis to a
patient) are also contemplated in the treatment methods of the
invention as discussed above.
[0070] In another aspect of the present invention, a kit is
provided for practicing the methods of the present invention. The
kit may include a carrier for the various components of the kit.
The carrier can be a container or support, in the form of, e.g.,
bag, box, tube, rack, and is optionally compartmentalized. The
carrier may define an enclosed confinement for safety purposes
during shipment and storage.
[0071] The kit also includes at least one component useful in
determining the status of exosomes and/or an exosomes-associated
marker using any of the detection techniques discussed herein. For
example, the kit many include probes to exosome-associated markers
(e.g., anti-CD63 antibodies) or probes to a panel of markers useful
for differentiating tissue or cancer types.
[0072] Various other components useful in the detection techniques
may also be included in the detection kit of this invention.
Examples of such components include, but are not limited to, Taq
polymerase, deoxyribonucleotides, dideoxyribonucleotides other
primers suitable for the amplification of a target DNA sequence,
RNase A, and the like. In addition, the detection kit preferably
includes instructions on using the kit for practicing the methods
of the present invention using human samples.
EXAMPLES
Example 1
Detection/Quantitation/Characterization of Exosomes by Flow
Cytometry
[0073] Exosomes may also be measured by flow cytometry. Flow
cytometry is generally used to measure cell-surface antigen
expression and thus may be readily adapted to measuring antigens on
the surface of exosomes. In general, exosomes are isolated from a
sample (e.g., serum) and then incubated with a labeled antibody
(e.g., fluorescence-labeled) against some surface marker expected
to be found on the exosomes (e.g., EpCAM). This solution is then
passed through a cell sorter, which detects and "counts" the number
of labeled exosomes as they flow through an optical/electronic
detection apparatus. Multiple labeled antibodies may be used in the
same sample as cell sorters are capable of detecting multiple
wavelengths of light at once. Flow cytometry may also be used to
detect and quantitate cytoplasmic antigens. For this it is
generally necessary to fix and permeabilize cells to enable
antibodies to gain access to them, after which the process is
generally the same as above.
[0074] The experiment detailed below has demonstrated that
capturing exosomes using anti-CD63 or anti-EpCAM antibodies and
quantitating them using flow cytometry can differentiate ovarian
cancer (OVCA) patients from non-cancerous controls.
[0075] Plasma samples were obtained from eight patients with no
cancer and from eight OVCA patients. These plasma samples were
subjected to differential centrifugation to enrich for exosomes as
follows and then protein was quantified by the Bradford Assay
(Bradford, A Rapid and Sensitive Method for the Quantitation of
Microgram Quantities of Protein Utilizing the Principle of
Protein-Dye Binding, ANAL. BIOCHEM. (1976) 72:248-254): [0076] 1.
Spun at 2000.times.g for 30 minutes--transferred supernatant to new
tube (contained exosomes) [0077] 2. Spun at 12000.times.g for 45
minutes--transferred supernatant to new tube (contained exosomes)
[0078] 3. Spun at 110,000.times.g for 2 hours
(ultracentrifugation)--pellet contained the exosomes [0079] 4.
Resuspended pellet in 1 ml PBS [0080] 5. Filtered through 0.2 .mu.m
filter [0081] 6. Spun at 110,000.times.g for 1 hour [0082] 7.
Resuspended in 100 .mu.l PBS
TABLE-US-00002 [0082] TABLE 2 mg/ml by Bradford .mu.l for 14 .mu.g
PBS OVCA Sample # 29449 1.09 12.8 87.2 31182 0.79 17.7 82.3 31216
0.77 18.2 81.8 32455 0.61 23.0 77.0 38704 0.62 22.6 77.4 39004 0.14
100.0 0.0 41365 0.44 31.8 68.2 41757 0.83 16.9 83.1 Normal Sample #
39270 1.49 9.4 90.6 39271 1.21 11.6 88.4 42201 0.79 17.7 82.3 42204
1.01 13.9 86.1 42205 0.75 18.7 81.3 42208 0.45 31.1 68.9 42209 0.54
25.9 74.1 42214 0.36 38.9 61.1
[0083] Latex beads were coated with anti-CD63 antibodies and then
contacted/incubated with the exosome samples isolated above as
follows: [0084] 1. Incubated 2.0 .mu.g of BD Pharmingen anti-CD63
antibody with 5 .mu.l Aldehyde latex bead 15 minutes at room
temperature. [0085] 2. Brought volume up to 500 .mu.l with PBS.
[0086] 3. Incubated overnight at 4.degree. C. with Rotor. [0087] 4.
Added 550 .mu.l 1M glycine in PBS and incubate 30 minutes at room
temperature. [0088] 5. Washed 3 times with PBS/0.5% BSA. [0089] 6.
Incubated with 14 .mu.g plasma pellet for 2 hours at room
temperature. [0090] 7. Washed 3 times with PBS/0.5% BSA. [0091] 8.
Resuspended in 250 .mu.l PBS/0.5% BSA.
[0092] The resulting solution was then incubated with phycoerythrin
(PE)-conjugated anti-CD63 antibody (CD63 BD Pharmingen Clone #H5C6)
and finally run through an Accuri C6 cell-sorter set to detect PE
(488 nm laser [excitation] and 585/40 nm filter for PE). Isotype
control samples comprising beads with PE-conjugated IgG1 antibodies
were also run through the cell sorter under identical conditions.
The fluorescence values (Median FL2-A) for these samples are shown
in Table 3 below:
TABLE-US-00003 TABLE 3 Plasma Exosome PE-Isotype Coated PE-cd63
(IgG1) Cell Latex Bead 0.013 mg/ml 0.2 mg/ml Staining Median Well
(10 .mu.l) Bdpharmigen BDpharmingen Buffer FL2-A 1 29449 20 .mu.l
50 .mu.l 128 2 31182 20 .mu.l 50 .mu.l 190 3 31216 20 .mu.l 50
.mu.l 128 4 32455 20 .mu.l 50 .mu.l 372 5 38704 20 .mu.l 50 .mu.l
263 6 39004 20 .mu.l 50 .mu.l 147 7 41365 20 .mu.l 50 .mu.l 526 8
41757 20 .mu.l 50 .mu.l 132 9 39270 20 .mu.l 50 .mu.l 125 10 39271
20 .mu.l 50 .mu.l 127 11 42201 20 .mu.l 50 .mu.l 137 12 42204 20
.mu.l 50 .mu.l 124 13 42205 20 .mu.l 50 .mu.l 128 14 42208 20 .mu.l
50 .mu.l 128 15 42209 20 .mu.l 50 .mu.l 127 16 42214 20 .mu.l 50
.mu.l 137 1 29449 1.3 .mu.l 68.7 .mu.l 120 2 31182 1.3 .mu.l 68.7
.mu.l 121 3 31216 1.3 .mu.l 68.7 .mu.l 122 4 32455 1.3 .mu.l 68.7
.mu.l 119 5 38704 1.3 .mu.l 68.7 .mu.l 118 6 39004 1.3 .mu.l 68.7
.mu.l 121 7 41365 1.3 .mu.l 68.7 .mu.l 120 8 41757 1.3 .mu.l 68.7
.mu.l 120 9 39270 1.3 .mu.l 68.7 .mu.l 123 10 39271 1.3 .mu.l 68.7
.mu.l 123 11 42201 1.3 .mu.l 68.7 .mu.l 119 12 42204 1.3 .mu.l 68.7
.mu.l 120 13 42205 1.3 .mu.l 68.7 .mu.l 118 14 42208 1.3 .mu.l 68.7
.mu.l 123 15 42209 1.3 .mu.l 68.7 .mu.l 124 16 42214 1.3 .mu.l 68.7
.mu.l 120
[0093] As seen in FIG. 3A, fluorescence was shifted substantially
higher in 4 out of 8 OVCA patients (31182, 32455, 38704, and
41365), with some shift in an additional two OVCA samples (39004,
41757). In contrast, in FIG. 3B no significant shift was seen in
any of the non-cancer controls (39270, 39271, 42201, 42204, 42205,
42208, 42209, and 42214).
Example 2
Detection/Quantitation/Characterization of Exosomes by ELISA
[0094] ELISA (Enzyme-Linked immunosorbent assay) can be adapted to
detect, quantify, and characterize exosomes according to the
invention. The purpose of an ELISA is to determine if a particular
protein is present in a sample and optionally, how much.
EpCAM-bearing exosomes were detected and quantitated in the
supernatant of epithelial cancer cell lines in the following
experiment.
[0095] Reagents: [0096] 1. Capture antibody is a mouse monoclonal
(clone 158210, R&D systems catalog #MAB 960) raised against the
extracellular domain of Human EpCAM. [0097] 2. Detection antibody
is a goat polyclonal (R&D systems catalog #BAF960) raised
against recombinant extracellular domain of Human EpCAM. [0098] 3.
Quantification achieved by comparison with a standard curve
generated using recombinant extracellular domain of Human EpCAM
(recombinant EpCAM/Fc chimera, R&D catalog #960-EP-050).
[0099] Sample Preparation: [0100] 1. Exosomes were isolated by
differential centrifugation and filtration using the same process
described in Example 1. [0101] 2. Pellets from final
ultracentrifuge spin were resuspended in PBS and measured for total
protein using the Bradford assay prior to addition to ELISA assay.
[0102] 3. Cell culture supernatants and supernatants from
ultracentrifugation were added directly to 96-well plates
pre-coated with the capture antibody for the assay: [0103] a.
Detection antibody was conjugated to biotin. [0104] b. Detection
enzyme was Horseradish-peroxidase conjugated to streptavidin
(R&D cat#DY998). [0105] c. Enzyme substrate was
H2O2+tetramethylbenzidine (R&D cat#DY999). [0106] d. The ELISA
was read on a Packard Bioscience Fusion microplate reader set at
450 nM.
[0107] Experiment:
[0108] Four epithelial cancer cell lines (SKOV-3, OVCa-5, MCF-7,
and HCT-15) have been evaluated by ELISA for presence of
EpCAM-bearing exosomes. EpCAM was measured by ELISA (1) in the
Cell-conditioned media and (2) in the Cell-conditioned media
depleted by centrifugation, and (3) total protein was also measured
in the centrifugation pellet. Results are summarized in Table 4
below:
TABLE-US-00004 TABLE 4 SKOV-3 OVCa-5 MCF-7 HCT-15 Medium 314 pg/ml
4,256 pg/ml 20,672 pg/ml 27,824 pg/ml [EpCAM] Depleted None
measured 4,160 pg/ml 1,921 pg/ml 3,016 pg/ml [EpCAM] Pellet 204
pg/ml 1022 pg/ml 9,616 pg/ml 14,032 pg/ml [EpCAM] Pellet 851
.mu.g/ml 1022 .mu.g/ml 882 .mu.g/ml 1,178 .mu.g/ml total protein
Pellet EP/ 0.65 0.24 0.46 0.5 medium EP g EpCAM/ 4.8 .times.
10.sup.-6 2.4 .times. 10.sup.-5 1.92 .times. 10.sup.-4 2.8 .times.
10.sup.-4 g total protein
[0109] If further analysis of the interior or contents of the
exosome is desired (such as by antibodies against cytoplasmic
proteins), the addition of formaldehyde followed by detergent will
permeabilize the exosome membranes. Thus the detection antibodies
may bind to interior exosome-associated antigens.
Example 3
Detection/Quantitation/Characterization of Exosomes by IHC
[0110] Exosome IHC will generally be carried out on an exosome
block. Exosome blocks are prepared by isolating exosomes from a
patient sample (e.g., plasma sample), fixing the exosome pellet
(e.g., with formalin), and then embedding the fixed pellet in some
medium (e.g., paraffin) that allows for convenient long-term
storage. Qualitative and quantitative IHC analysis may then be
performed on thin slices of the exosome blocks using antibodies
against proteins of interest on the exterior or interior of the
exosomes.
Example 4
Detection/Quantitation/Characterization of Exosomes by ECL
ELISA
[0111] A variation of ELISA that may be used in the methods of the
invention employs electrochemiluminescence (ECL). ECL ELISA is
described in detail in, e.g., PCT Application Publication No.
WO/1987/006706. Briefly, a sample is contacted with a detection
antibody (e.g., anti-CD63) labeled with an electrochemiluminescent
chemical moiety, the resulting sample is exposed to electrochemical
energy, and the electromagnetic radiation emitted by the
electrochemiluminescent chemical moiety is detected. The chief
advantage of such an approach is the improved signal to noise ratio
achieved using ECL. ECL can be extended beyond immunological assays
to nucleic acid probe-based assays.
[0112] ECL ELISA was used to differentiate the plasma of cancer
patients from the plasma of healthy controls. Plasma samples were
obtained and diluted with 1% MSD Blocker A, without any initial
enrichment for exosomes. The plasma samples used were as follows:
47 prostate cancer, 49 breast cancer, 48 colorectal cancer, 48 lung
cancer, 50 ovarian cancer, and 70 healthy controls.
[0113] The Meso Scale Discovery.RTM. ("MSD") platform was used in
the following ECL ELISA experiments. MSD 96 well High Bind plates
were incubated with a capture antibody (BD CD63--4 .mu.g/ml) or
protein (.alpha.V.beta.6 integrin--0.2 .mu.g/ml) overnight. The
plate was then washed three times with 3000 Wash Buffer (PBS 0.01%
Tween). The plate was then passivated with 1% MSD Blocker A for 1
hour on an orbital shaker. The plate was then washed three times
with 300 .mu.l Wash Buffer. Diluted plasma samples were then
allowed to incubate in the plate for 2 hours at room temperature on
an orbital shaker. The plate was then washed three times with 300
.mu.l Wash Buffer. 25 .mu.l of the detection antibody was then
loaded into each well and this was allowed to incubate for 1 hour
at room temperature on an orbital shaker. The plate was then washed
three times with 300 .mu.l wash buffer.
[0114] The following antibodies were used:
TABLE-US-00005 TABLE 5 Antibody Target Vendor Cat. # CD63 BD
Biosciences 556019 Caudin-3 R&D Systems mab4620 Claudin-4
R&D Systems mab4219 CD36 USBiological c2388-04b HLA-DR
eBioscience 13-9956-82 EpCAM R&D Systems 842008 .alpha.V.beta.6
integrin R&D Systems 3817-AV CD147 AbCAM AB666
In cases where the detection antibody had been directly labeled
with the SULFO-TAG conjugate, 150 .mu.l of 1.times. Read Buffer T
(cat#R92TD-2) was added and the resulting sample was read on the
MSD SECTOR Imager 6000. In cases where the antibody had not been
not directly labeled the appropriate secondary antibody, e.g.,
SULFO-TAG Streptavidin, was used for detection. The secondary
antibody was allowed to incubate for 30 minutes at room temperature
on an orbital shaker. The plate was then washed three times with
300 .mu.l Wash Buffer. 150 .mu.l of 1.times. Read Buffer T
(cat#R92TD-2) was added and the resulting sample was read on the
MSD SECTOR Imager 6000.
[0115] The various capture-detection pairs differentiated cancers
from controls as follows:
TABLE-US-00006 TABLE 6 Disease Marker Pair p-value Breast
.alpha.V.beta.6 integrin Capture -- CD63 Detect 2.90E-13 Breast
CD63 Capture -- CD36 Detect 4.17E-12 Breast CD63 Capture -- CD147
Detect 4.65E-08 Breast CD63 Capture -- Claudin-3 Detect 2.65E-06
Breast CD63 Capture -- HLA-DR Detect 6.00E-04 Breast
.alpha.V.beta.6 integrin Capture -- CD147 Detect 1.27E-03 Breast
CD63 Capture -- EpCAM Detect 2.99E-02 Breast CD63 Capture --
Claudin-4 Detect 8.42E-02 Colon/Rectal .alpha.V.beta.6 integrin
Capture -- CD63 Detect 3.26E-22 Colon/Rectal CD63 Capture -- CD36
Detect 2.93E-19 Colon/Rectal CD63 Capture -- CD147 Detect 8.20E-14
Colon/Rectal .alpha.V.beta.6 integrin Capture -- CD147 Detect
1.50E-09 Colon/Rectal CD63 Capture -- HLA-DR Detect 2.66E-07
Colon/Rectal CD63 Capture -- Claudin-3 Detect 2.71E-04 Colon/Rectal
CD63 Capture -- EpCAM Detect 3.19E-02 Colon/Rectal CD63 Capture --
Claudin-4 Detect 3.87E-02 Lung CD63 Capture -- CD147 Detect
3.20E-03 Lung .alpha.V.beta.6 integrin Capture -- CD63 Detect
3.35E-03 Lung CD63 Capture -- CD36 Detect 5.57E-03 Lung
.alpha.V.beta.6 integrin Capture -- CD147 Detect 1.02E-01 Lung CD63
Capture -- HLA-DR Detect 1.47E-01 Lung CD63 Capture -- Claudin-3
Detect 7.07E-01 Lung CD63 Capture -- Claudin-4 Detect 8.08E-01 Lung
CD63 Capture -- EpCAM Detect 8.26E-01 Ovarian CD63 Capture --
Claudin-3 Detect 2.98E-09 Ovarian CD63 Capture -- CD36 Detect
2.00E-08 Ovarian .alpha.V.beta.6 integrin Capture -- CD63 Detect
5.05E-08 Ovarian CD63 Capture -- HLA-DR Detect 5.42E-07 Ovarian
.alpha.V.beta.6 integrin Capture -- CD147 Detect 3.44E-04 Ovarian
CD63 Capture -- CD147 Detect 4.01E-04 Ovarian CD63 Capture -- EpCAM
Detect 2.30E-02 Ovarian CD63 Capture -- Claudin-4 Detect 8.12E-01
Prostate CD63 Capture -- CD36 Detect 2.25E-07 Prostate CD63 Capture
-- CD147 Detect 7.56E-07 Prostate CD63 Capture -- Claudin-3 Detect
7.38E-06 Prostate .alpha.V.beta.6 integrin Capture -- CD63 Detect
1.89E-05 Prostate .alpha.V.beta.6 integrin Capture -- CD147 Detect
1.99E-04 Prostate CD63 Capture -- Claudin-4 Detect 8.11E-04
Prostate CD63 Capture -- HLA-DR Detect 6.52E-03 Prostate CD63
Capture -- EpCAM Detect 1.41E-02
[0116] These correlations generally held up under multivariate
analysis. See FIG. 6. Specifically, for CD36, Claudin-3 and
Claudin-4:
TABLE-US-00007 TABLE 7 Disease p-value AUC Breast 1.70E-12 0.88
Lung 4.00E-03 0.72 Colon/Rectal 3.70E-19 0.95 Ovarian 1.40E-08 0.81
Prostate 9.80E-08 0.81
[0117] These results are summarized in FIG. 5. Thus the
capture-detection pairs above can detect prostate, ovarian, lung,
colorectal, and breast cancer in the plasma of patients.
Example 5
Detection/Quantitation/Characterization of Exosomes by ECL ELISA
after Exosome Enrichment
[0118] ECL ELISA was combined with differential centrifugation to
separate ovarian cancer patients from controls. Differential
centrifugation was performed as described in Example 1 above. ECL
ELISA was performed on the resulting enriched samples essentially
as described in Example 5 above, except that enriched exosome
samples were directly captured on the MSD plate rather than on an
MSD plate coated with a capture reagent. Specifically, 25 .mu.l of
each sample was the allowed to absorb onto an MSD 96 well High Bind
Plate (Cat #L11XB-6). The plate was then passivated using MSD
Blocker A (Cat #R93AA-1). The resulting plate was then incubated
with SULFO-TAG labeled anti-CD63 antibody (CD63 BD Pharmingen Clone
#H5C6) and read on a MSD SECTOR Imager 6000. The results are
summarized in Table 8, below, and FIG. 7.
TABLE-US-00008 TABLE 8 Sample Name Signal 256031 4970 256030 8872
256029 8127 256028 3624 H4522 593
[0119] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be clear to those skilled in the art that
certain changes and modifications may be practiced within the scope
of the appended claims.
* * * * *